Human diploid fibroblasts undergo a limited number of population doublings when grown in tissue culture. The number of doublings is inversely proportional to the age of the donor. Hayflick and Moorehead (1961) have suggested that this limited proliferation potential is an indication of aging at the cellular level. Bell et al. (1978, 1979) have suggested the division characteristics are indicative of a differentiation of the cells as in wound healing in the organism. Fibroblasts in wounds differentiate into myofibroblasts and function in wound contraction. The rate of wound contraction decreases in older individuals, causing extended recovery and hospitalization. The specific aim of this proposal is to test the hypothesis that fibroblasts leave cycle in culture and differentiate into a state resembling the myofibroblast. We concentrate on changes in the structure and function of the cytoskeleton, the organelle system of the fibroblast which functions in motility and contraction. We will compare non-dividing fibroblasts from young and old donors and early and late passage cultures to establish changes in cytoskeletal morphology and function. Cells will be evaluated for 1) the conversion of cytoskeletal elements into stress fibers as revealed by light and electron microscopy; 2) the calcium requirements of stress fiber contractility induced by ATP in glycerin-extracted cell models; and 3) the contractile abilities of fibroblasts cultured in three-dimensional collagen lattices as measured by the rate of gel shrinkage in days and the tension produced when the cells are stimulated to contract with agonists known to produce tension in strips of granulation tissue. Knowledge gained from these experiments will aid us in evaluating the effects of aging in the organism and aging in culture on the contractile abilities of human diploid fibroblasts.